Androsta-1,4-diene-17-carbothioic acid, 6,9-difluoro-11,17-dihydroxy-16-methyl-3-oxo-, (6.alpha.,11.beta.,16.alpha.,17.alpha.)-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Thio-Acid

IUPAC Name:

Thio-Acid

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Thio-Acid
- Substance type: Powder
- Physical state: Solid
- Analytical purity: 98.75% area (based on impurities)
- Lot/batch No.: 0000120575

Specific details on test material used for the study:

Identification: Thio Acid
Physical state/Appearance: White powder
Batch: 0000120575
Purity: 98.76%
Expiry date: 5 July 2016
Storage conditions: Room temperature in the dark
Intended use/Application: Isoloated chemical intermediate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The test samples were thawed with the aid of sonication. The 72-Hour test samples were centrifuged (4500 rpm, 10 minutes) to remove algal cells, then all the test samples were diluted (if required) with test medium. See Table 1 for more information on preparation volumes.
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Test solutions

Vehicle:

yes

Remarks:

The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

Details on test solutions:

An amount of test item (1100 mg) was dispersed, in duplicate, in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. The test item was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 48 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded) to give a nominal test concentration of approximately 118 mg/L. Measured concentrations of 75 mg/L and 67 mg/L were obtained from the 100% v/v saturated solution prepared for use in the range-finding and definitive test respectively. These results were much closer to those obtained from the other pre-treatment types at 48 hours suggesting that the measured concentration of 118 mg/L was erroneous.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Strain: CCAP 278/4.
Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of
approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 - 105 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

No remarks

Post exposure observation period:

Not mentioned

Test conditions

Hardness:

Not mentioned

Test temperature:

24 ± 1 ºC throughout the test

pH:

8.1 at 0 h to 8.2 at 72 h

Dissolved oxygen:

Not mentioned

Salinity:

Not mentioned

Conductivity:

Not mentioned

Nominal and measured concentrations:

Nominal concentrations: 1, 3.2, 10, 32 and 100 % v/v saturated solution

Details on test conditions:

The pH value of the control cultures (see Table 2) was observed to increase from pH 8.1 at 0 hours to pH 8.2 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines. Temperature was maintained at 24 ± 1 ºC throughout the test.

Reference substance (positive control):

yes

Remarks:

A positive control (Study Number 41501180) used potassium dichromate as the reference item. The positive control was conducted between 12 May 2015 and 15 May 2015.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 43 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 46 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 58 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 6.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 9

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 10

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 58 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized
interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line. It was not possible to calculate 95% confidence limits for the EC50 values as the data generated did not fit the models available for the calculation of confidence limits.
There were no statistically significant differences between the control, 0.47, 1.7, 5.8 and 18 mg/L test concentrations (P≥0.05), however the 58 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 18 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 58 mg/L.
Statistical analysis of the yield data showed that there were no statistically significant differences between the control, 0.47, 1.7, 5.8 and 18 mg/L test concentrations (P≥0.05), however the 58 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 18 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 58 mg/L.

Results with reference substance (positive control):

The following data show that the cell concentration of the control cultures increased by a factor of 147 after 72 hours. This increase was in line with the OECD Guideline that states the
enhancement must be at least by a factor of 16 after 72 hours.
Mean cell density of control at 0 hours : 4.85 x 103 cells per mL
Mean cell density of control at 72 hours : 7.10 x 105 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 9% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.
growth rate NOEC: 0.25 mg/l
yield NOEC: 0.25 mg/l
growth rate LOEC: 0.50 mg/l
yield LOEC: 0.50 mg/l

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Any other information on results incl. tables

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.47, 1.7, 5.8 and 18 mg/L, however no intact cells were observed to be present in the test cultures at 58 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:
Growth rate: EC50 : 53 mg/l, NOEC : 18 mg/l, LOEC : 58 mg/l
Yield: EC50 : 35 mg/l, NOEC : 18 mg/l, LOEC : 58 mg/l

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods

Preliminary solubility work conducted indicated that the test item was not readily soluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 118 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to precondition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.56 to 67 mg/L. A decline in measured test concentration was observed at 72 hours to between 0.39 and 49 mg/L (67% to 78% of the 0-Hour measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Response variable	EC50 (mg/l)	NOEC (mg/l)	LOEC (mg/l)
Growth rate	53	18	58
Yield	35	18	58